Mitochondrial oxidative phosphorylation (OXPHOS) is a critical process for energy metabolism, and the primary source of cellular adenosine triphosphate (ATP). OXPHOS takes place at the mitochondrial respiratory chain (MRC), which is localized at the inner mitochondrial membrane and comprises five enzyme complexes (complex I to V) and two mobile electron carriers (ubiquinone and cytochrome c) [1]. Quantitative and functional deficiencies of the MRC have been implicated in a wide range of health and disease states, including mitochondrial disorders [2], neurodegenerative diseases such as Parkinson’s disease (PD) [3, 4], Alzheimer’s disease (AD) [5], and amyotrophic lateral sclerosis (ALS) [6], as well as in aging [7, 8]. Given its broad relevance, the development and application of reliable methods to detect and assess MRC deficiencies in post-mortem brain tissue is of paramount importance.

MRC is commonly assessed by quantitative methods, such as immunohistochemistry (IHC) and immunoblotting, and functional measurements of specific complex activities [9,10,11,12]. Interpretation of bulk tissue data can be complicated, given that mitochondrial abundance and MRC complex stoichiometry can vary significantly across different cell types, complicating the interpretation of bulk tissue data. On the other hand, IHC is a more cumbersome, low-throughput method, both in execution and interpretation, but offers the unique advantage of identifying changes at the single-cell level. IHC has been extensively used to map MRC complexes across different tissues, including the brain in both mitochondrial and neurodegenerative disorders [6, 13], as well as in aging [14, 15].

Traditionally, IHC of brain tissue has been performed to detect a single antigen at a time. Staining for MRC enzymes and mitochondrial markers has typically been conducted on sequential sections from the same brain region, using horseradish peroxidase (HRP) with the brown substrate 3,3’-diaminobenzidine (DAB) [11, 16], and analysis of stained sections involves manually counting DAB-positive and -negative neurons from digital images. However, identifying negative neurons can be challenging, as their cytoplasm lacks distinct color, leading to potential uncertainty in interpretation. The lack of co-staining for a mitochondrial marker also makes it difficult to know whether regional variations in mitochondrial mass contribute to differences in staining.

To address this limitation, introducing a second mitochondrial antigen, such as VDAC1, stained with a different color would enhance the accuracy of identifying neurons lacking specific MRC complexes. In recent years, a magenta-colored HRP substrate has been described, offering compatibility with automated staining protocols and the ability to be combined with DAB staining [17]. Building on these advancements, we aimed to adapt and validate a sequential double-staining method using DAB and magenta chromogens for quantitative measurements of MRC complexes in human brain tissue. To develop and validate this approach, we used samples from patients with POLG-related disease. This disease model is particularly well-suited for validating the double-staining chromogen method, as it exhibits a characteristic profile of MRC deficiencies: specifically, deficiencies in complexes I and IV while sparing complex II [13].